Neuropeptides are important modulators of nervous system function from worms to humans. Produced by specialized neurosecretory cells, their controlled release into local brain circuits and the circulatory system has profound effects on chemosensation, feeding, circadian rhythms, sleep, social behavior, and general physiological homeostasis. In Aedes aegypti mosquitoes, neuropeptides have been implicated in the cyclical behavioral suppression of host attraction that lasts for three days after the female has taken a blood-meal. Although sugar- feeding is sufficient for survival, once female mosquitoes reach reproductive maturity, they require a blood-meal to develop eggs. Attraction to human host cues, including body heat, carbon dioxide, body odor, and taste cues on skin, is only expressed when the female needs blood protein for egg production. For approximately 72 hours after a blood-meal, female attraction to humans is suppressed. This cycle repeats up to 10 times for the approximately one month adult life-span. The spreading of infectious diseases among humans is a by- product of this blood-feeding cycle, so elucidating the mechanisms of host suppression has great health relevance. The broad, long-term objectives of this research are to gain a comprehensive understanding of mosquito neuropeptide and neuropeptide receptor function and to define the mechanisms that mediate the strong suppression of host-seeking behavior after a blood-meal. The proposal has three interlocking specific aims: (1) Annotate neuropeptide and neuropeptide receptor genes in Aedes aegypti and define ligand-receptor interactions. (2) Identify neuropeptide signaling pathways that mediate host-seeking suppression after a blood-meal. (3) Develop a genetic tool-kit for studying neuropeptide signaling in the mosquito. The work is innovative because it uses high-throughput 384-well cell-based screening with a genetically-encoded calcium indicator to deorphanize neuropeptide receptors; pharmacological and neurogenetic tools to probe mechanisms underlying post blood-meal host-suppression; and optimized CRISPR/Cas9 genome-editing to generate a large collection of mutant strains, each lacking a given neuropeptide or neuropeptide receptor. The project will yield significant insights into the ligands and biological function of neuropeptide receptors in this important disease vector insect and will provide new tools to regulate mosquito host-seeking behavior. The strains developed under this project will be a valuable community resource for investigating the physiological functions of neuropeptide signaling.